The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison of the aa sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved aa in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa. The probable function of these domains is discussed. Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).
Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA-1470 and toxB-1470. A polymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VP10463, the toxB-1470 gene has 16 additional nucleotides, 13 within the 5'-untranslated region and three within the coding region. The M(r) of ToxB-1470 is 269,262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are M(r) 269,709 and IP 4.13. In comparison with ToxB, ToxB-1470 differs primarily in the N-terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C-terminal region between residues 869-2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB-1470 with ToxA and ToxB reveals the highest homology between ToxB-1470 and ToxB. Thus ToxB-1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic effects of ToxB-1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii, but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or alpha-toxin (Tcn alpha) of Clostridium novyi. The LT-like action of ToxB-1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.
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